The present invention is directed to oligonucleotides containing C3xe2x80x2-methylene hydrogen phosphonate monomers and methods for the preparation of such monomers and oligonucleotides.
It is well known that most of the bodily states in mammals, including most disease states, are effected by proteins. Proteins, either acting-directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man.
Classical therapeutics generally has focused upon interactions with proteins in an effort to moderate their disease-causing or disease-potentiating functions. Recently, however, attempts have been made to moderate the production of proteins by interactions with the molecules (i.e., intracellular RNA) that direct their synthesis. These interactions have involved hybridization of complementary xe2x80x9cantisensexe2x80x9d oligonucleotides or certain analogs thereof to RNA. Hybridization is the sequence-specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to RNA or to single stranded DNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects.
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. The pharmacological activity of antisense oligonucleotides and oligonucleotide analogs, like other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor for oligonucleotides is the stability of the species in the presence of nucleases. It is unlikely that unmodified oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases. Modification of oligonucleotides to render them resistant to nucleases therefore is greatly desired.
Modification of oligonucleotides to enhance nuclease resistance generally has taken place on the phosphorus atom of the sugar-phosphate backbone. Examples of such modifications include methyl phosphonate, phosphorothioate, phosphorodithioate, phosphoramidate and phosphotriester linkages, and 2xe2x80x2-O-methyl ribose sugar units. Phosphate-modified oligonucleotides, however, generally have suffered from inferior hybridization properties. See, e.g., Cohen, J. S., ed. Oligonucleotides: Antisense Inhibitors of Gene Expression, (CRC Press, Inc., Boca Raton, Fla., 1989). Other modifications to oligonucleotides include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
Another key factor is the ability of antisense compounds to traverse the plasma membrane of specific cells involved in the disease process. Cellular membranes consist of lipid-protein bilayers that are freely permeable to small, nonionic, lipophilic compounds and are inherently impermeable to most natural metabolites and therapeutic agents. See, e.g., Wilson, Ann. Rev. Biochem. 1978, 47, 933. The biological and antiviral effects of natural and modified oligonucleotides in cultured mammalian cells have been well documented. It appears that these agents can penetrate membranes to reach their intracellular targets. Uptake of antisense compounds into a variety of mammalian cells, including HL-60, Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells has been studied using natural oligonucleotides and certain nuclease resistant analogs, such as alkyl triesters and methyl phosphonates. See, e.g., Miller et al., Biochemistry 1977, 16, 1988; Marcus-Sekura et al., Nuc. Acids Res. 1987, 15, 5749; and Loke et al., Top. Microbiol. Immunol. 1988, 141, 282.
Often, modified oligonucleotides and oligonucleotide analogs are internalized less readily than their natural counterparts. As a result, the activity of many previously available antisense oligonucleotides has not been sufficient for practical therapeutic, research or diagnostic purposes. Two other serious deficiencies of prior art compounds designed for antisense therapeutics are inferior hybridization to intracellular RNA and the lack of a defined chemical or enzyme-mediated event to terminate essential RNA functions.
Modifications to enhance the effectiveness of the antisense oligonucleotides and overcome these problems have taken many forms. These modifications include base ring modifications, sugar moiety modifications and sugar-phosphate backbone modifications. Prior sugar-phosphate backbone modifications, particularly on the phosphorus atom, have effected various levels of resistance to nucleases. However, while the ability of an antisense oligonucleotide to bind to specific DNA or RNA with fidelity is fundamental to antisense methodology, modified phosphorus oligonucleotides have generally suffered from inferior hybridization properties.
Replacement of the phosphorus atom has been an alternative approach in attempting to avoid the problems associated with modification on the pro-chiral phosphate moiety. For example, Matteucci, Tetrahedron Letters 1990, 31, 2385 discloses the replacement of the phosphorus atom with a methylene group. However, this replacement yielded unstable compounds with nonuniform insertion of formacetal linkages throughout their backbones. Cormier et al., Nucleic Acids Research 1988, 16, 4583, discloses replacement of phosphorus with a diisopropylsilyl moiety to yield homopolymers having poor solubility and hybridization properties. Stirchak et al., Journal of Organic Chemistry 1987, 52, 4202 discloses replacement of phosphorus linkages by short homopolymers containing carbamate or morpholino linkages to yield compounds having poor solubility and hybridization properties. Mazur et al., Tetrahedron 1984, 40, 3949, discloses replacement of a phosphorus linkage with a phosphonic linkage yielding a homotrimer. Goodchild, Bioconjugate Chemistry 1990, 1, 165, discloses ester linkages that are enzymatically degraded by esterases and, therefore, are not suitable for antisense applications.
Phosphonates are known in the literature but very few 3xe2x80x2-hydrogen phosphonates of nucleosides are known. Synthesis and use of 3xe2x80x2-methylene phosphonic acids and their esters as isosteres of nucleoside-3xe2x80x2-phosphates and phosphodiesters have been reported. Albrecht et al., Tetrahedron, 1984, 40, 79; Jones et al., J. Am. Chem. Soc., 1970, 5510; Albrecht et al., J. Am. Chem. Soc., 1970, 5511. All of these phosphonic acids and phosphonates bear 2xe2x80x2-hydroxy groups. Related 2xe2x80x2-hydroxy-3xe2x80x2-phosphonate analogs of nucleoside phosphates have been synthesized through a multistep synthetic procedure. Morr et al., Z. Naturforsch., 1983, 38b, 1665; Morr and Ernst, Liebigs Ann. Chemie, 1993, 1205. 2xe2x80x2-Deoxy-thymidine-3xe2x80x2-methylene-hydrogen phosphonates have also been reported in WO97/32887 (Sep. 12, 1997).
Thus far only thymidine-3xe2x80x2-methylene hydrogen phosphonates have been reported. Furthermore, these phosphonates bear t-butyldiphenylsilyl protecting groups on the 5xe2x80x2-position which makes use of such phosphonates in conventional oligonucleotide synthesis cumbersome. There exists a need to provide isosteric 3xe2x80x2-methylene hydrogen phosphonate nucleosides that can bear a variety of substitutents at the 2xe2x80x2- and 5xe2x80x2-positions.
Phosphonates have been investigated because of their isosteric relationship with the natural phosphodiester linkage of oligonucleotides. Methyl phosphonates, in which one of the non-nucleosidic oxygen atoms of the natural phospodiester linkage is replaced with a methyl group, have been studied widely. Related isosteres, where either the 3xe2x80x2- or the 5xe2x80x2-oxygen atom of the phosphodiester linkage is replaced with a methylene group, have also been investigated. Mazur et al. have reported the synthesis of a 3xe2x80x2-methylenephosphonate isostere of UpUpU (Tetrahedron, 1984, 40, 3949). This oligonucleotide is limited by the presence of 2xe2x80x2-hydroxy groups on all residues and by the tedious synthesis of the sugar-methylenephosphonate prior to attachment of a nucleosidic base. More recently, Heinemann et al. have reported the synthesis of 2xe2x80x2-deoxy-3xe2x80x2-methylenephosponate oligonucleotides and the effects of such a structural change on the conformation of an A-DNA octamer double helix (Nucleic Acids Research, 19, 427). 2xe2x80x2-Deoxy-thymidine-3xe2x80x2-methylene-hydrogen phosphonates have also been reported in WO97/32887 (published Sep. 12, 1997). Isosteric 5xe2x80x2-methylenephosphonate oligonucleotides have also been synthesized and reported to bind to complementary nucleic acids and be resistant to phosphodiesterases (Breaker et al., Biochemistry, 1993, 32, 9125).
Early attempts to synthesize phosphonate isosteres of nucleoside phosphates commenced with conversion of diacetone-D-glucose, through a series of ten reactions, into uridine-3xe2x80x2-methylene phosphonate protected at the 2-xe2x80x2and 5xe2x80x2-positions with hydroxyl groups (Mazur et al., Tetrahedron, 1984, 40, 3949). The key transformations entailed an Arbuzov reaction using triisopropyl phosphite to generate a ribofuranose-3xe2x80x2-methylenephosphonate, which was subsequently reacted with 1,2-bis(trimethylsiloxy)uracil and stannous chloride to afford the protected uridine-3xe2x80x2-methylene phosphonate.
More recently, Novartis has reported a multi-step procedure for the synthesis of 2xe2x80x2-deoxy-thymidine-3xe2x80x2-methylene hydrogen phosphonates (WO97/32887, published Sep. 12, 1997). Synthesis commences with 5xe2x80x2-protected thymidine (Derry et al., Anti-Cancer Drug Design, 1993, 8, 203; Mihkailov et al., Nucleosides and Nucleotides, 1996, 15, 1323), which is thiocarbonylated to afford the 5xe2x80x2-O-TBDPS-T-3xe2x80x2-thiocarbonate, using 3-t-butylphenoxy chlorothionoformate (Sanghvi et al., Synthesis, 1994, 1163). This thiocarbonate is reacted with tributylstannylstyrene (PhCHxe2x95x90CHSnBu3) in the presence of AIBN, followed by oxidation of the 3xe2x80x2-alkenyl intermediate so generated using osmium tetroxide and sodium periodate, to afford 5xe2x80x2-O-TBDPS-T-3xe2x80x2-aldehyde (Lebreton et al., Tetrahedron Letters, 1994, 35, 5225; Sanghvi et al., Synthesis, 1994, 1163). Reduction of the 3xe2x80x2-aldehyde to the 3xe2x80x2-methanol derivative, followed by iodination using a phosphonium iodide afforded the key 5xe2x80x2-O-TBDPS-T-3xe2x80x2-methyl iodide intermediate (WO97/32887, published Sep. 12, 1997). This iodide was then subjected to a six-step sequence of reactions to convert the 3xe2x80x2-methyl iodide into the 3xe2x80x2-methylene hydrogen phosphonate group. The sequence consisted of sequential reactions with potassium hexamethyldisilazide and a H-phosphinate reagent, trimethylsilyl chloride for three days, titanium tetraisopropoxide, acetic acid and TBAF to deprotect the TBDPS group, tritylation using DMT-chloride, and finally hydrolysis using methanol/sodium methoxide or DBU. However, this monomer synthesis is lengthy and cumbersome, and has been proven effective for the synthesis of only 2xe2x80x2-deoxy-thymidine nucleosides.
Antisense therapy needs modified oligomers and oligonucleotides that bear a variety of nucleoside residues and diversity of modifications. A method for the convenient and efficient synthesis of a variety of nucleoside-3xe2x80x2-methylene hydrogen phosphonates is therefore needed. Further, these monomers need to be readily adaptable in oligonucleotide synthesis protocols.
Despite interest in isosteric oligonucleotides and methylenephosphonate oligomers, there has been no report of the successful synthesis and use of 2xe2x80x2-modified-3xe2x80x2-methylenephosphonate oligonucleotides capable of enhanced binding to complementary nucleic acids and exhibiting increased resistance to phosphodiesterases.
The limitations of available methods for modification of the phosphorus backbone have led to a continuing and long-felt need for other modifications which provide resistance to nucleases and satisfactory hybridization properties for antisense oligonucleotide diagnostics and therapeutics.
The present invention provides monomers having formula I: 
wherein:
Bx is a protected or unprotected heterocyclic base moiety;
T1 is an oligonucleotide, oligonucleoside, nucleoside, nucleotide, H or a hydroxyl protecting group;
X1 is O, S or NH;
R1 is Z0xe2x80x94(C2-C20 alkynyl);
R2 is H, F, Cl, Br, I, alkyl, substituted alkyl, (CH2)nxe2x80x94OCH3, (CH2)nNH2 or (CH2)a(Q1)b(Q2)c;
or R1 has one of formulas XI or XII: 
xe2x80x83wherein:
Z0 is O, S or NH;
E is C1-Cl10 alkyl, N(Q1)(Q2) or Nxe2x95x90C(Q1)(Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support;
or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
q1 is from 1 to 10;
q2 is from 1 to 10;
q3 is zero or 1;
q4 is zero, 1 or 2;
q5 is 1 to 10;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z4 is OM1, SM1, or N(M1)2;
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1)(Q2), OQ1, halo, SQ1 or CN;
Z6 is OH, OCH2CH2CN, N(i-Pr)2, dialkylamino, disubstituted alkylamino or Oxe2x88x92HBy+;
By is an organic base moiety;
each of Y1 and Y2 is, independently, H, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cyano, carboxyl, ester or a cyclic moiety; and
m is 0 or 1.
The present invention also provides oligomers of formula II: 
wherein:
Bx is a protected or unprotected heterocyclic base moiety;
T1 is an oligonucleotide, oligonucleoside, nucleoside, nucleotide, H or a hydroxyl protecting group;
each of X1, X2 and X3 is, independently, O, S or NH;
L is C(Y1)(Y2) or O, provided that at least one L is C(Y1)(Y2);
R1 is Z0xe2x80x94(C2-C20 alkynyl);
or R1 has one of formulas XI or XII: 
xe2x80x83wherein:
Z0 is O, S or NH;
E is C1-C10 alkyl, N(Q1)(Q2) or Nxe2x95x90C(Q1)(Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support;
or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
q1 is from 1 to 10;
q2 is from 1 to 10;
q3 is zero or 1;
q4 is zero, 1 or 2;
q5 is 1 to 10;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z4 is OM1, SM1, or N(M1)2;
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1)(Q2), OQ1, halo, SQ1 or CN;
m is 0 or 1;
p is 0 or an integer from 1 to 50; and
each of Y1 and Y2 is, independently, H, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cyano, carboxyl, ester or a cyclic moiety.
The present invention further provides methods of preparing monomers of formula I: 
wherein:
Bx is a protected or unprotected heterocyclic base moiety;
T1 is an oligonucleotide, oligonucleoside, nucleoside, nucleotide, H or a hydroxyl protecting group;
X1 is O, S or NH;
R is Z0xe2x80x94(C2-C20 alkynyl);
or R1 has one of formulas XI or XII: 
xe2x80x83wherein:
Z0 is O, S or NH;
E is C1-C10 alkyl, N(Q1)(Q2) or Nxe2x95x90C(Q1)(Q2);
each Q1 and Q2 is, independently, H, C1-Cl10 alkyl, substituted alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support;
or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
q1 is from 1 to 10;
q2 is from 1 to 10;
q3 is zero or 1;
q4 is zero, 1 or 2;
q5 is 1 to 10;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z4 is OM1, SM1, or N(M1)2;
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1)(Q2), OQ1, halo, SQ1 or CN;
Z6 is OH, OCH2CH2CN, N(i-Pr)2, dialkylamino, disubstituted alkylamino or Oxe2x88x92HBy+;
m is 0 or 1;
each of Y1 and Y2 is, independently, H, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cyano, carboxyl, ester or a cyclic moiety; and
By is an organic base moiety; comprising the steps of:
(a) providing a 5xe2x80x2-protected-3xe2x80x2-substituted alkyl nucleoside of formula III: 
xe2x80x83wherein
xe2x80x83X is a leaving group;
(b) reacting the nucleoside of formula III with bis(trimethylsilyl)phosphonite;
(c) concentrating the product of step (b);
(d) adding methanolic solvent to the product of step (c) to form a solution; and
(e) concentrating said solution.
The present invention further provides methods of preparing monomers of formula I: 
wherein:
Bx is a protected or unprotected heterocyclic base moiety;
T1 is an oligonucleotide, oligonucleoside, nucleoside, nucleotide, H or a hydroxyl protecting group;
X1 is O, S or NH;
R1 is Z0xe2x80x94(C2-C20 alkynyl);
or R1 has one of formulas XI or XII: 
xe2x80x83wherein:
Z0 is O, S or NH;
E is C1-C10 alkyl, N(Q1)(Q2) or Nxe2x95x90C(Q1)(Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support;
or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
q1 is from 1 to 10;
q2 is from 1 to 10;
q3 is zero or 1;
q4 is zero, 1 or 2;
q5 is 1 to 10;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z4 is OM1, SM1, or N(M1)2;
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1)(Q2), OQ1, halo, SQ1 or CN;
Z6 is OH, OCH2CH2CN, N(i-Pr)2, dialkylamino, disubstituted alkylamino or Oxe2x88x92HBy+;
m is 0 or 1;
each of Y1 and Y2 is, independently, H, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cyano, carboxyl, ester or a cyclic moiety; and
By is an organic base moiety; comprising the steps of:
(a) providing a 5xe2x80x2-protected-3xe2x80x2-hydroxy alkyl nucleoside of formula IV: 
(b) oxidizing said nucleoside to form an oxidized nucleoside;
(c) alkenylating said oxidized nucleoside with a Wittig reagent to form a 3xe2x80x2-substituted nucleoside;
(d) phosphonylating said 3xe2x80x2-substituted nucleoside; and
(e) protecting the 5xe2x80x2-hydroxy group of said 3xe2x80x2-substituted nucleoside.
Also provided are methods for preparing oligomers of formula II: 
wherein:
Bx is a protected or unprotected heterocyclic base moiety;
T1 is an oligonucleotide, oligonucleoside, nucleoside, nucleotide, H or a hydroxyl protecting group;
each of X1, X2 and X3 is, independently, O, S or NH;
L is C(Y1)(Y2) or O, provided that at least one L is C(Y1)(Y2);
R1 is Z0xe2x80x94(C2-C20 alkynyl);
or R1 has one of formulas XI or XII: 
xe2x80x83wherein:
Z0 is O, S or NH;
E is C1-C10 alkyl, N(Q1)(Q2) or Nxe2x95x90C(Q1)(Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support;
or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
q1 is from 1 to 10;
q2 is from 1 to 10;
q3 is zero or 1;
q4 is zero, 1 or 2;
q5 is 1 to 10;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z4 is OM1, SM1, or N(M1)2;
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1)(Q2), OQ1, halo, SQ1 or CN;
m is 0 or 1;
p is 0 or an integer from 1 to 50; and
each of Y1 and Y2 is, independently, H, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cyano, carboxyl, ester or a cyclic moiety; comprising the steps of:
(a) providing a 5xe2x80x2-protected nucleoside attached to a solid support;
(b) cleaving the 5xe2x80x2-protecting group in the presence of an acid solution to form a 5xe2x80x2-deprotected nucleoside;
(c) coupling said 5xe2x80x2-deprotected nucleoside with a monomer of formula I: 
xe2x80x83wherein:
Z6 is OH, OCH2CH2CN, N(i-Pr)2, dialkylamino, disubstituted alkylamino or Oxe2x88x92HBy+;
each of Y1 and Y2 is, independently, H, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cyano, carboxyl, ester or a cyclic moiety; and
By is an organic base moiety; in the presence of a condensing agent to form a compound having an H-phosphonate linkage;
(d) optionally capping unreacted 5xe2x80x2-hydroxy groups using isopropyl phosphite and pivaloyl chloride in acetonitrile and pyridine;
(e) repeating steps (b) to (d) to form an oligomer of desired length and composition;
(f) oxidizing said H-phosphonate linkage with an oxidizing reagent to form an oxidized methylenephosphonate linkage;
(g) repeating steps (b) to (f) to form an oligomer of desired length and composition; and
(h) cleaving said oligomer with aqueous ammonia.
In a preferred embodiment the condensing reagent is a solution of 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane in an organic solvent and the oxidizing reagent is a mixture of camphorsulfonyloxaziridine (CSO) and N,O-bis(trimethylsilyl)acetamide in an organic solvent.